The measurement of carnitine and acyl-carnitines: application to the investigation of patients with suspected inherited disorders of mitochondrial fatty acid oxidation.

We describe an improved radio-enzymatic method for the measurement of carnitine, short-chain acyl-carnitine and long-chain acyl-carnitine in plasma and tissue. An internal standard, hexadecanoyl-[CH3-3H]-carnitine was synthesised and used to improve the determination of long-chain acyl-carnitine. The between and within batch precisions were 10.4 and 7%, respectively. Control data for neonates, infants, children and adults in the fed and fasted state are documented. In addition we confirm the hypocarnitinaemia associated with pregnancy. Patients with medium-chain acyl-CoA dehydrogenase deficiency were studied during episodes of hypoglycaemia. In both fasted controls and patients there were high concentrations of short-chain acyl-carnitine, however in the latter group there were also low concentrations of free carnitine. We suggest that the monitoring of plasma carnitine and its derivatives is a useful adjunct to the investigation of children suspected to suffer from inherited disorders of mitochondrial beta-oxidation. We also describe a sample preparation procedure suitable for high performance liquid chromatographic analysis of specific acyl-carnitines from urine, plasma and tissue homogenates. The recoveries of acetyl-carnitine, octanoyl-carnitine and hexadecanoyl carnitine from urine were 101.5, 95 and 91% and from plasma 99.5, 91.5 and 85.5%, respectively. Acyl-carnitines (C2-C16) were analysed as their p-bromophenacyl derivatives by reverse-phase high performance liquid chromatography using a ternary gradient of acetonitrile/water/triethylamine phosphate. We report ten patients who excreted octanoyl-carnitine, hexanoyl-carnitine and in some cases a small amount of decanoyl-carnitine. In most of these cases suberylglycine and dicarboxylic acids were also detected by GC/MS. We had access to cultured fibroblasts from five of these patients and were able to demonstrate medium-chain acyl-CoA dehydrogenase deficiency by direct enzyme assay.     

Validation of the survey of work styles: a profile measure of the type A behaviour pattern

The present study compares the Type A classification accuracy of the Jenkins Activity Survey (JAS), The Framingham Type A Scale, and a new Type A behaviour pattern (TABP) measure. The Survey of Work Styles (SWS), a self-report measure of the TABP, was developed using a construct approach to scale construction. It consists of six content scales. Impatience, Anger, Work Involvement, Time Urgency, Job Dissatisfaction and Competitiveness. In addition to the six content scales, a seventh scale, Scale A, is comprised of items empirically selected to relate to the Rosenman Structured Interview. In the present study the SWS was found to be significantly related to both the JAS, and the Framingham Type A Scale in a sample of 163 business managers. Median reliability of the SWS subscales was 0.82, and for the total scale 0.90. Discriminant function analysis using cross validational jackknifing procedures resulted in a classification accuracy of 83% of the Type A managers in relation to the Structured Interview. Classification using the SWS was found to correlate significantly higher with the Structured Interview than did either classification with the JAS or with the Framingham Type A Scale. Modal profile analysis yielded three independent bipolar typal dimensions, indicating that a single dimension or classification of the TABP represents an oversimplification of a complex behaviour pattern. These results support the reconceptualization of the TABP in terms of distinct facets and profile patterns.     

NIFEDIPINE BLOCKS ACTH AND CORTISOL RELEASE IN MAN

1. The present study investigated the effect of prior administration of nifedipine on AVP-induced ACTH release in seven normal volunteers. Three protocols were used: 20 mg oral nifedipine; 0.14 pressor units intramuscular (i.m.) per kg bodyweight aqueous AVP; oral nifedipine plus i.m. AVP 90 min later. Plasma ACTH and cortisol were measured at intervals for 2.5 h during each test. 2. The mean peak plasma ACTH and cortisol levels and the mean peak changes from basal in these levels were significantly lower in the nifedipine/AVP test than in the AVP alone test. The integrated area under the cortisol time curve was significantly lower for the nifedipine/AVP test than that for the AVP test alone. Nifedipine alone caused no changes in ACTH or cortisol. 3. Acute administration of oral nifedipine caused an inhibition of AVP-stimulated ACTH and cortisol release in normal humans. This effect may be due to blockade of plasma membrane calcium channels normally activated during AVP stimulation of pituitary corticotrophs.     

Bioavailability of estradiol as a marker for breast cancer risk assessment

The search for a hormonal marker in breast cancer has centered on estrogens and their metabolites. However, direct measurements of total amounts of these steroids have shown no convincing or consistent differences between normal women and women with breast cancer. The purpose of this study was to measure the percentages of non-protein-bound estradiol (%NPBE) and of estradiol bound to albumin (%ABE) and the levels of sex hormone-binding globulin (SHBG) both in women with breast cancer and in those free of disease. Serum was collected and analyzed within 2 weeks, using an isodialysis method. The mean %NPBE and %ABE were significantly higher in 32 women with breast cancer (1.73 and 64.0%, respectively) than in 32 matched disease-free women (1.43 and 48.6%, respectively) (P less than 0.001). No significant difference was observed in the levels of plasma albumin when the above matched groups were compared. However, plasma levels of SHBG were significantly lower in the women with breast cancer than in either the control population or matched controls. In this finding we differ from previous studies which reported no significant differences in the mean plasma levels of SHBG. In our study, the increased %NPBE and %ABE in some patients with breast cancer may be related to a lower level of plasma SHBG; other factors, too, may affect the distribution of estradiol. Our results support the hypothesis than an increase in %NPBE and %ABE or both may indicate an increased risk of breast cancer.     

Evaluation of bruises and areas of induration after two techniques of subcutaneous heparin injection

The subcutaneous administration of the anticoagulant heparin sodium is a frequently performed nursing intervention. Bruising (discoloration) and induration (hardening) occur after some but not all such injections. This has implications for nursing; not only does the patient experience the physical discomfort and the psychologic impact of visible body trauma, but bruising and induration limit possible sites for future injections. Administration technique is frequently cited as a possible cause of bruising and induration. The purpose of this study was to compare two administration techniques currently being used by nurses. Variables studied included syringe size, change of needles after drawing medication into the syringe, use of an air bubble, and type of sponge (dry or alcohol) applied to the site after injection. The sample included 50 medical-surgical patients aged 23 to 88 years. Each subject received two injections by the same investigator using two different techniques. Sites were inspected and bruises and induration measured 52 hours after each injection. To compare the size of bruises and indurations, the data were analyzed by the Mann-Whitney U-Wilcoxon rank sum test, which showed a 0.003 level of significance for bruises and a 0.02 level of significance for induration. To compare the number of subjects in whom bruises and indurations developed, the data were analyzed by the chi-square test, which showed a 0.0458 level of significance for induration but only a 0.1371 level of significance for bruising.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular forms of gonadotropin-releasing hormone associated peptide (GAP): changes within the rat hypothalamus and release from hypothalamic cells in vitro

We have developed RIAs using antisera directed against the cryptic peptide of the GnRH precursor (termed GnRH-associated peptide, GAP) and have used these together with a GnRH assay to characterize proGnRH-derived peptides in rat hypothalamic extracts. On Sephadex chromatography we have identified three molecular forms of GAP-like immunoreactivity (GAP-LI), in addition to the GnRH decapeptide. The largest of these forms is an 8.0-kilodalton (kD) GAP-LI which appears to be the complete proGnRH peptide. The second is a 6.5-kD GAP-LI, and is similar to the complete cryptic peptide (i.e. proGnRH14-69 or GAP1.56). The third peptide is a 2.5 kD C-terminal fragment of the cryptic peptide, representing a processed form of GAP. In whole hypothalamic extracts from normal rats the 8.0-kD form was the major form, comprising 60-70% of the total GAP-LI. All three forms were present in three distinct areas of the rat hypothalamus, namely median eminence (ME), anterior and mid-hypothalamus. However in the ME the proportion of 8.0-kD GAP-LI was significantly reduced and the proportion of 6.5-kD GAP-LI significantly increased compared to anterior and mid-hypothalamic samples (p less than 0.05). In whole hypothalamic extracts from pregnant and lactating rats the total content of proGnRH-derived peptides was reduced but the relative proportions of these peptides were not significantly changed from normal female rats. However, in postlactating rats, 2 weeks after removal of pups, the total levels of GAP-LI were unchanged compared to normals, but the percentage of 8.0-kD GAP-LI was significantly decreased and the percentage of 2.5-kD GAP-LI significantly increased compared to normals (p less than 0.05), suggesting that proGnRH may undergo additional processing dependent on physiological condition. In fetal and neonatal rats the proportion of the 6.5-kD peptide was increased and that of the 8.0-kD peptide decreased compared to adults, and this change became less significant with increasing age. In ovariectomized rats the proportion of 6.5-kD GAP-LI was increased and that of 8.0-kD GAP-LI decreased; this change was partially reversed with steroid treatment. Both the 6.5 and 2.5-kD forms were released by high K+ stimulation of neonatal hypothalamic cells in culture. These results indicate that there is differential processing of the proGnRH precursor within the hypothalamus and in altered physiological states.     

Post-translational processing of thyrotropin-releasing hormone precursor in rat brain: identification of 3 novel peptides derived from pro TRH

The sequence of rat hypothalamic pro-thyrotropin releasing hormone, deduced by sequencing of cDNA, in addition to 5 TRH progenitor genitor sequences contains leader, trailer and 4 intervening sequences separated by paired basic amino acid sequences. We have developed radioimmunoassays to synthetic peptides corresponding to portions of these cryptic proTRH sequences and have used these assays to identify and partially characterize proTRH peptides, distinct from TRH, in extracts of rat brain. Two of these peptides correspond closely in size to one intervening sequence and the car☐y-terminal sequence of proTRH. Three other peptides correspond to the intact amino-terminal leader sequence and two peptides formed by a further cleavage of the leader sequence at an internal paired basic amino acid sequence.